Cytoskeletal-driven membrane dynamics requires the concerted action of microtubules, actin, and their associated motors and activators. Although specific motors and adaptors have been identified that participate in the transport, tubulation, and retraction of biological membranes, the molecular mechanisms of many of these processes are not well defined. This gap in knowledge is due, in part, to the lack of understanding of the biophysical parameters of the key cytoskeletal components that drive these dynamic membrane events. Knowledge of these parameters allows formation of quantitative models that consider the mechanical and kinetic limits of protein function. Therefore, this subproject proposes to use single-molecule and ensemble biophysical techniques to measure directly the motile and mechanical properties of cytoplasmic dynein (mammalian and yeast) and kinesin (KIF16B) motors. Results will be correlated directly with new insights from analysis of motor structural dynamics obtained in collaboration with Dr. Goldman, and will provide the biophysical parameters required to better understand the cellular and in vitro experiments performed collaboration with Dr. Holzbaur. We will also determine mechanical properties of the key scaffolding proteins that link membranes, motors, and cytoskeletal filaments. These interactions include the attachment of KIF16B to phosphoinositides, the dynein-bicaudalD-Rab6-membrane complex, and the interaction of WHAMM with membranes. The results will be correlated directly to biochemical, structural, and cellular experiments performed in collaboration with Drs. Dominguez and Holzbaur. Our studies will provide the biophysical parameters required to generate quantitative models for the roles of cytoskeletal motors and scaffolding protein in powering membrane dynamics and transport.